Pegylated fluorobenzamide analogues, their synthesis and use in diagnostic imaging

ABSTRACT

Pegylated fluoroalkoxybenzamide compounds which selectively bind Sigma-2 receptors are disclosed. These compounds, when labeled with a positron-emitting radioisotope such as  18 F, can be used as radiotracers for medical imaging such as imaging of tumors by positron emission tomography (PET). In addition, these compounds, when labeled with  123 I, can be used as radiotracers for imaging of tumors by single photon emission computed tomography (SPECT). Methods for synthesis of these compounds are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalApplication Ser. No. 61/429,758, filed Jan. 5, 2011, which isincorporated herein by reference in its entirety.

INTRODUCTION

Sigma receptors are a class of receptors that are expressed in manynormal tissues, including liver, kidneys, endocrine glands, and thecentral nervous system (CNS) (Walker, J. M., et al. Pharmacol Rev 42:355-402 1990). It has been well established that there are at least twotypes of sigma receptors, sigma-1 (σ₁) and sigma-2 (σ₂) (Walker, J. M.,et al. Pharmacol Rev 42, 355-402, 1990). Overexpression of σ₂ receptorshas been reported in a variety of human and murine tumors (Bem, W. T.,et al., Cancer Res 51: 6558-6562, 1991; Vilner, B. J., et al., In:Multiple sigma and PCP receptor ligands: mechanisms for neuromodulationand neuroprotection?, Kamenka, J. M., and Domino, E. F., ed, Ann Arbor(Mich.), 7 NPP Books, p. 341-353, 1992; Mach, R. H., et al., Cancer Res.57: 156-161, 1997).

Searches for σ₂ selective ligands has led to the identification of anumber compounds having modest to high selectivity for σ₂ versus σ₁receptors. As set forth in FIG. 1, these compounds include CB-184 (10),CB-64D (11), BIMU-1 (12) (Bowen, W. D., et al., Eur. J. Pharmacol. 278:257-260, 1995; Bonhaus, D. W., et al., J. Pharmacol. Exp. Ther. 267:961-70, 1993). and PB-167 (13) (Colabufo, N. A., et al., J. Pharm.Pharmacol. 57: 1453-1459, 2005; Kassiou, M., et al., Bioorg. Med. Chem.13: 3623-3626, 2005; Berardi, F., et al., J. Med. Chem. 2004, 47:2308-2317) as well as certain benzamide analogs (14-16) (Mach, R. H., etal., Bioorg. Med. Chem. 11: 225-233, 2003; Huang, Y., et al., J. Med.Chem. 44: 4404-15, 2001; U.S. patent application Ser. No. 10/903,771 toMach et al). We previously reported the evaluation of several ¹¹C, ⁷⁶Brand ^(125/123)I radiolabelled conformationally-flexible benzamideanalogs using EMT-6 tumor-bearing female Balb/c mice (Tu, Z., et al.,Nucl. Med. Biol. 32: 423-430, 2005; Xu, J., et al., Eur. J. Pharmacol.525 (1-3): 8-17, 2005; Hou, C., et al., Nucl. Med. Biol. February, 33:203-9, 2006). Initial in vivo studies of5-methyl-2-[¹¹C]-methoxy-N-[2-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl]-benzamideand5-[⁷⁶Br]-bromo-2,3-dimethoxy-N-[2-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl]-benzamideindicated that these compounds were potential radiopharmaceuticals forimaging solid tumors and their proliferative status with positronemission tomography (PET). However, the radionuclide properties of ⁷⁶Brand ¹¹C make these isotopes less than ideal for PET imaging. Forexample, images produced by PET using ⁷⁶Br as a radiotracer are oftenblurry, (Laforest, R., et al., IEEE Transactions on Nuclear Science, 49:2119-2126, 2002), and the short half-life of ¹¹C (t_(1/2)=20.4 min)places time constraints on tracer synthesis and duration of scansessions. Contrast between tumor and normal tissues can be less thansatisfactory when a σ₂-selective radiotracer tagged with ¹¹C is used inPET imaging.

U.S. Pat. No. 7,659,400 to Mach et al., discloses compounds and saltsthereof of structure

wherein m is an integer from 1 to about 10, n is an integer from 1 toabout 10, and R₁ and R₂ are each independently selected from the groupconsisting of H, a halogen selected from the group consisting of I, Br,Cl and F, a C₁₋₄ alkoxy, a C₁₋₄ alkyl, a C₁₋₄ fluoroalkyl, a C₁₋₄fluoroaloxy, CF₃, OCF₃, SCH₃, SCF₃, and NH₂. In some embodiments, the Fcan be an ¹⁸F. In some embodiments, R₁ or R₂ can be an iodine (I), whichcan be an ¹²³I. However, some of these compounds may not have thesolubility, clearance rate, toxicity or stability that may be desirablefor some uses. Accordingly, alternative σ₂-selective ligands that can beused as radiotracers in PET imaging are needed.

SUMMARY

The present inventors have developed a series of compounds. In someembodiments, a radiolabeled compound or salt thereof disclosed hereincan be used as a tracer for diagnostic imaging, such as diagnosticimaging of tumors. In various configurations, a compound or salt thereofdisclosed herein can be used as a tracer in imaging methods such aspositron emission tomography (PET) or single photon emission computedtomography (SPECT). In various embodiments, a compound or salt thereofof the present teachings can selectively bind Sigma receptors, and inparticular can bind Sigma-2 receptors in preference to Sigma-1receptors. In various embodiments, the compounds and salts thereof canalso selectively bind to tumor cells, and thus can also be used astracers for detecting tumor cells. In some embodiments, a compound orsalt thereof can comprise a positron-emitting radioisotope such ¹¹C,¹³N, ¹⁵O, ¹⁸F, ⁷⁶Br or ¹²⁴I. In various configurations, such compoundsand salts thereof can be used as radiotracers for imaging biologicaltissue such as tumors using SPECT. In some embodiments, a compound orsalt thereof can comprise a gamma (γ)-emitting radioisotope such as¹²³I.

In some embodiments, a compound of the present teachings is a pegylatedfluoroalkoxybenzamide compound or a salt thereof having a structure

wherein m is an integer from 2 to 5, n is an integer from 1 to about 10,and X₁ and X₂ are each independently selected from the group consistingof H, a halogen selected from the group consisting of I, Br, Cl and F, aC₁₋₄ alkoxy, a C₁₋₄ alkyl, a C₁₋₄ fluoroalkyl, a C₁₋₄ fluoroalkoxy, CF₃,OCF₃, SCH₃, SCF₃, and NH₂. In some embodiments, m can be 2. In someembodiments, m can be 3. In some embodiments, m can be 4. In someembodiments, m can be 5 In some embodiments, n can be 1. In someembodiments, n can be 2. In some embodiments, n can be 3. In someembodiments, n can be 4. In some embodiments, n can be 5. In someembodiments, n can be 6. In some embodiments, X₁ can be H. In someembodiments, X₁ can be methoxy (OCH₃). In some embodiment, X₂ can bemethyl (CH₃). In some embodiment, X₂ can be a halogen selected from F,Cl, Br, and I. In some embodiments, X₂ can be bromine (Br). In someembodiments, X₂ can be iodine (I). In some embodiments, m=2 or 3, n=4,X₁=H or OCH₃, and X₂=CH₃, Br or I. In various embodiments, an I can be a¹²³I or a ¹²⁴I.

In some embodiments, a compound or salt thereof of the present teachingscan include at least one ¹⁸F isotope. A compound or a salt thereof ofthese embodiments can be a pegylated radiolabelled fluoroalkoxybenzamidecompound or a salt thereof having a structure

wherein m is an integer from 2 to 5, n is an integer from 1 to about 10,and X₁ and X₂ are each independently selected from the group consistingof H, a halogen selected from the group consisting of I, Br, Cl and F, aC₁₋₄ alkoxy, a C₁₋₄ alkyl, a C₁₋₄ fluoroalkyl, a C₁₋₄ fluoroalkoxy, CF₃,OCF₃, SCH₃, SCF₃, and NH₂. In some embodiments, m can be 2. In someembodiments, m can be 3. In some embodiments, m can be 4. In someembodiments, m can be 5 In some embodiments, n can be 1. In someembodiments, n can be 2. In some embodiments, n can be 3. In someembodiments, n can be 4. In some embodiments, n can be 5. In someembodiments, n can be 6. In some embodiments, X₁ can be H. In someembodiments, X₁ can be methoxy (OCH₃). In some embodiment, X₂ can bemethyl (CH₃). In some embodiment, X₂ can be a halogen selected from thegroup consisting of F, Cl, Br, and I. In some embodiments, X₂ can bebromine (Br). In some embodiments, X₂ can be iodine (I). In someembodiments, m=2 or 3, n=4, X₁=H or OCH₃, and X₂=CH₃, Br or I.

In some embodiments, a compound or salt thereof can have a structureselected from the group consisting of

a salt thereof,

a salt thereof,

a salt thereof,

a salt thereof,

a salt thereof,

a salt thereof,

a salt thereof,

and a salt thereof. In some embodiments, the fluorine atom comprised byany of these compounds or salts can be an ¹⁸F. In some embodiments, incompounds or salts thereof comprising bromine, such as 6c, 6d, 6g or 6h,the bromine can be a ⁷⁶Br. In some embodiments, in compounds or saltsthereof comprising iodine, such as 6e or 6f, the iodine can be an ¹²³I,an ¹²⁴I or an ¹³¹I.

Other embodiments of the present teachings include methods ofsynthesizing a pegylated fluoroalkoxybenzamide compound or salt thereofwherein the compound has structure

wherein m is an integer from 2 to 5, n is an integer from 1 to about 10,and X₁ and X₂ are each independently selected from the group consistingof H, a halogen selected from the group consisting of I, Br, Cl and F, aC₁₋₄ alkoxy, a C₁₋₄ alkyl, a C₁₋₄ fluoroalkyl, a C₁₋₄ fluoroalkoxy, CF₃,OCF₃, SCH₃, SCF₃, and NH₂. In some embodiments, m can be 2. In someembodiments, m can be 3. In some embodiments, m can be 4. In someembodiments, m can be 5 In some embodiments, n can be 1. In someembodiments, n can be 2. In some embodiments, n can be 3. In someembodiments, n can be 4. In some embodiments, n can be 5. In someembodiments, n can be 6. In some embodiments, X₁ can be H. In someembodiments, X₁ can be methoxy (OCH₃). In some embodiment, X₂ can bemethyl (CH₃). In some embodiment, X₁ can be a halogen selected from F,Cl, Br, and I. In some embodiments, X₂ can be bromine (Br). In someembodiments, X₂ can be iodine (I). In some embodiments, m=2 or 3, n=4,X₁=H or OCH₃, and X₂=CH₃, Br or I.

Syntheses of various fluoropegylated compounds of the present teachingsare outlined in scheme 1 and scheme 2. Scheme 1: Commercially-available6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (1) was converted into4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butanenitrile (2) byN-alkylation with 4-bromobutanenitrile using triethylamine as base.Reduction of intermediate 2 with lithium alumina hydride intetrahydrofuran gave4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butan-1-amine (3) Thissynthetic route to compound 3 was published previously (Tu et al. NuclMed Biol, 2005. 32(5): p. 423-430). The target fluoropegylated compoundswere made using the three step sequence shown in Scheme 2. First, thesubstituted salicylic acids were condensed with the intermediate 3 usingN,N′-Dicyclohexylcarbodiimide (DCC) as the coupling agent to give thesalicylamides 4a-d. Second, O-alkylation of the phenol group in 4a-dwith diethylene glycol or triethylene glycol with potassium carbonate asbase afforded benzamides 5a-h. Third, the fluoropegylated benzamidesprepared by treatment of 5a-h with Diethylaminosulfur trifluoride (DAST)to give the target compounds, 6a-h.

The present disclosure includes the following aspects:

-   1. A pegylated fluoroalkoxybenzamide compound or salt thereof of    structure

wherein m is an integer from 2 to 5, n is an integer from 1 to 10, andX₁ and X₂ are each independently selected from the group consisting ofH, a halogen selected from the group consisting of I, Br, Cl and F, aC₁₋₄ alkoxy, a C₁₋₄ alkyl, a C₁₋₄ fluoroalkyl, a C₁₋₄ fluoroalkoxy, CF₃,OCF₃, SCH₃, SCF₃, and NH₂.

-   2. A pegylated fluoroalkoxybenzamide compound or salt thereof in    accordance with aspect 1, wherein m=2, n=4, X₁ is H, and X₂ is CH₃.-   3. A pegylated fluoroalkoxybenzamide compound or salt thereof in    accordance with aspect 1, wherein m=3, n=4, X₁ is H, and X₂ is CH₃.-   4. A pegylated fluoroalkoxybenzamide compound or salt thereof in    accordance with aspect 1, wherein m=2, n=4, X₁ is H, and X₂ is Br.-   5. A pegylated fluoroalkoxybenzamide compound or salt thereof in    accordance with aspect 1, wherein m=3, n=4, X₁ is H, and X₂ is Br.-   6. A pegylated fluoroalkoxybenzamide compound or salt thereof in    accordance with aspect 1, wherein m=2, n=4, X₁ is H, and X₂ is I.-   7. A pegylated fluoroalkoxybenzamide compound or salt thereof in    accordance with aspect 1, wherein m=3, n=4, X₁ is H, and X₂ is I.-   8. A pegylated fluoroalkoxybenzamide compound or salt thereof in    accordance with aspect 1, wherein m=2, n=4, X₁ is methoxy, and X₂ is    Br.-   9. A pegylated fluoroalkoxybenzamide compound or salt thereof in    accordance with aspect 1, wherein m=3, n=4, X₁ is methoxy, and X₂ is    Br.-   10. A radiolabeled pegylated fluoroalkoxybenzamide compound or salt    thereof of structure

wherein m is an integer from 2 to 5, n is an integer from 1 to 10, andX₁ and X₂ are each independently selected from the group consisting ofH, a halogen selected from the group consisting of I, Br, Cl and F, aC₁₋₄ alkoxy, a C₁₋₄ alkyl, a C₁₋₄ fluoroalkyl, a C₁₋₄ fluoroalkoxy, CF₃,OCF₃, SCH₃, SCF₃, and NH₂.

-   11. A radiolabeled pegylated fluoroalkoxybenzamide compound or salt    thereof in accordance with aspect 10, wherein m=2, n=4, X₁ is H, and    X₂ is CH₃.-   12. A radiolabeled pegylated fluoroalkoxybenzamide compound or salt    thereof in accordance with aspect 10, wherein m=3, n=4, X₁ is H, and    X₂ is CH₃.-   13. A radiolabeled pegylated fluoroalkoxybenzamide compound or salt    thereof in accordance with aspect 10, wherein m=2, n=4, X₁ is H, and    X₂ is Br.-   14. A radiolabeled pegylated fluoroalkoxybenzamide compound or salt    thereof in accordance with aspect 10, wherein m=3, n=4, X₁ is H, and    X₂ is Br.-   15. A radiolabeled pegylated fluoroalkoxybenzamide compound or salt    thereof in accordance with aspect 10, wherein m=2, n=4, X₁ is H, and    X₂ is 1.-   16. A radiolabeled pegylated fluoroalkoxybenzamide compound or salt    thereof in accordance with aspect 10, wherein m=3, n=4, X₁ is H, and    X₂ is 1.-   17. A radiolabeled pegylated fluoroalkoxybenzamide compound or salt    thereof in accordance with aspect 10, wherein m=2, n=4, X₁ is    methoxy, and X₂ is Br.-   18. A radiolabeled pegylated fluoroalkoxybenzamide compound or salt    thereof in accordance with aspect 10, wherein m=3, n=4, X₁ is    methoxy, and X₂ is Br.-   19. A radiolabeled pegylated fluoroalkoxybenzamide compound or salt    thereof of structure

wherein m is an integer from 2 to 5, n is an integer from 1 to about 10,and X₁ is selected from the group consisting of H, a halogen selectedfrom the group consisting of I, Br, Cl and F, a C₁₋₄ alkoxy, a C₁₋₄alkyl, a C₁₋₄ fluoroalkyl, a C₁₋₄ fluoroalkoxy, CF₃, OCF₃, SCH₃, SCF₃,and NH₂ and X₂ is selected from the group consisting of ⁷⁶Br, ¹²³I and¹²⁴I.

-   20. A radiolabeled pegylated fluoroalkoxybenzamide compound or salt    thereof in accordance with aspect 19, wherein m=2, n=4, X₁ is H, and    X₂ is ⁷⁶Br.-   21. A radiolabeled pegylated fluoroalkoxybenzamide compound or salt    thereof in accordance with aspect 19, wherein m=3, n=4, X₁ is H, and    X₂ is ⁷⁶Br.-   22. A radiolabeled pegylated fluoroalkoxybenzamide compound or salt    thereof in accordance with aspect 19, wherein m=2, n=4, X₁ is H, and    X₂ is ¹²³I or ¹²⁴I.-   23. A radiolabeled pegylated fluoroalkoxybenzamide compound or salt    thereof in accordance with aspect 19, wherein m=3, n=4, X₁ is H, and    X₂ is ¹²³I or ¹²⁴I.-   24. A radiolabeled pegylated fluoroalkoxybenzamide compound or salt    thereof in accordance with aspect 19, wherein m=2, n=4, X₁ is    methoxy, and X₂ is ⁷⁶Br.-   25. A radiolabeled pegylated fluoroalkoxybenzamide compound or salt    thereof in accordance with aspect 19, wherein m=3, n=4, X₁ is    methoxy, and X₂ is ⁷⁶Br.-   26. A method of imaging a tumor in a mammal, the method comprising:

administering to the mammal a radiolabelled fluoroalkoxybenzamidecompound comprising a positron-emitting radionuclide of any one ofaspects 10-25, or a salt thereof; and

subjecting the mammal to positron emission tomography (PET) scanning,wherein the compound comprises a positron-emitting radionuclide.

-   27 A method of imaging a tumor in a mammal, the method comprising:

administering to the mammal a radiolabelled fluoroalkoxybenzamidecompound of any one of aspects 10-25, or a salt thereof; and

subjecting the mammal to single photon emission computed tomography(SPECT) scanning, wherein the compound comprises a gamma-emittingradionuclide.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 illustrates prior art compounds.

FIG. 2 illustrates tumor uptake of ¹⁸F-labeled pegylated analogs 6c, 6dand 6g versus a nonpegylated analog (m=1; X₁=H; X₂=Br).

DETAILED DESCRIPTION

The present inventors have developed a series of pegylated compoundswhich can be used as radiolabels for diagnostic imaging, in particularpositron emission tomography (PET) imaging of tumors. The compoundsselectively bind Sigma receptors, and in particular bind Sigma-2receptors in preference to Sigma-1 receptors. The compounds are alsobelieved to bind selectively to tumor cells, and thus can be used astracers for detecting tumor cells. Without being limited by theory, itis generally believed that many types of tumor cells have a high densityof sigma-2 receptors, and therefore compounds of the present teachingscan be effective tracers for detecting tumors by virtue of thecompounds' affinity for the sigma-2 receptors. In addition, because insome embodiments, the compounds comprise the radioisotope ¹⁸F, apreferred isotope for imaging by positron emission tomography (PET),they can be effective as radiotracers for PET imaging of tumors inhumans or other mammals. Furthermore, in some embodiments, the compoundscan comprise the gamma-emitting radioisotope ¹²³I, a preferred isotopefor imaging by single photon emission computed tomography (SPECT). Thesecompounds can be effective as radiotracers for SPECT imaging of tumorsin humans or other mammals.

The following non-limiting examples are provided to further illustratethe present teachings and are not intended to limit the scope of anyclaim. Unless specifically presented in the past tense, an example canbe a prophetic or an actual example. The examples are not intended tolimit the scope of the aspects. The methods described herein utilizelaboratory techniques well known to skilled artisans, and guidance canbe found in laboratory manuals and textbooks such as Sambrook, J., etal., Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 2001; Spector, D. L. et al.,Cells: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1998; and Harlow, E., Using Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1999; Hedrickson et al., Organic Chemistry 3rd edition,McGraw Hill, New York, 1970; Carruthers, W., and Coldham, L, ModernMethods of Organic Synthesis (4th Edition), Cambridge University Press,Cambridge, U.K., 2004; Curati, W. L., Imaging in Oncology, CambridgeUniversity Press, Cambridge, U.K., 1998; Welch, M. J., and Redvanly, C.S., eds. Handbook of Radiopharmaceuticals: Radiochemistry andApplications, J. Wiley, New York, 2003.

In the experiments described in herein, all reagents were purchased fromcommercial suppliers and used without further purification unlessotherwise stated. Synthetic intermediates were purchased from AldrichChemical Co. (Milwaukee, Wis.) and Lancaster Synthesis (Windham, Mass.)and used as received unless otherwise stated. Tetrahydrofuran (THF) wasdistilled from sodium hydride immediately prior to use. Anhydroustoluene was distilled from sodium/toluene shortly before use. Allanhydrous and all air-sensitive reactions were carried out in oven-driedglassware under an inert nitrogen atmosphere unless otherwise stated.Standard handling techniques for air sensitive materials were employedthroughout this study. When the reactions involve extraction withdichloromethane (CH₂Cl₂), chloroform (CHCl₃), ethyl acetate (EtOAc), orethyl ether (Et₂O), the organic solutions was dried with anhydrousNa₂SO₄ and concentrated with a rotary evaporator under reduced pressure.Flash column chromatography can be conducted using silica gel 60a, “40Micron Flash” [32-63 μm] (Scientific Adsorbents, Inc.). Melting pointscan be determined using the MEL-TEMP 3.0 apparatus and left uncorrected.¹H NMR spectra can be recorded at 300 MHz on a Varian Mercury-VXspectrometer with CDCl₃ as solvent and tetramethylsilane (TMS) as theinternal standard. All chemical shift values are reported in ppm.Elemental analyses (C, H, N) can be determined by Atlantic Microlab,Inc. Yields were not optimized. Melting points were determined on aHaake-Buchler or MeI-Temp melting point apparatus and are uncorrected.¹H NMR spectra were recorded at 300 MHz on a Varian Mercury-VXspectrometer with CDCl₃ as the solvent and tetramethylsilane (TMS) asthe internal standard. NMR spectra are referenced to the deuterium lockfrequency of the spectrometer. The chemical shifts (in ppm) of residualsolvents were observed at 7.26 (CHCl₃) or 4.78 (CD₃OH). The followingabbreviations were used to describe peak patterns wherever appropriate:b=broad, d=doublet, t=triplet, q=quartet, m=multiplet. Analytical thinlayer chromatography (TLC) was performed on Analtech GHLF silica gelglass plates, and visualization was aided by UV. Elemental analyses (C,H, N) were determined by Atlantic Microlab, Inc. The purity of thetarget compounds was determined by elemental analysis and by HPLCmethods. All pegylated compounds reported herein have a purity ≧95%.

Example 1

This example demonstrates General procedure A for synthesizing somecompounds of the present teachings.

N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-hydroxy-5-methylbenzamide(4a). A mixture of 5-methylsalicylic acid (0.691 g, 4.5 mmol),4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butan-1-amine (1.0 g,3.8 mmol), N,N′-Dicyclohexylcarbodiimide (DCC) (0.94 g, 4.5 mmol) and1-Hydroxybenzotriazole hydrate (HOBOt) (0.61 g, 4.5 mmol) indichloromethane (20 mL) was stirred overnight under an atmosphere ofnitrogen. The reaction mixture was washed with saturated sodiumbicarbonate solution (3×10 mL). The organic solution was dried overNa₂SO₄ and volatiles were removed under reduced pressure. The residuewas purified by silica gel column with methanol/ether (5/95, v/v) as themobile phase to afford 4a as a viscous oil (1.39 g; 93%). ¹H NMR (300MHz, CDCl₃): 1.70-1.78 (m, 4H), 2.09 (s, 3H), 2.50-2.60 (m, 2H),2.68-2.76 (m, 2H), 2.78-2.88 (m, 2H), 3.44-3.60 (m, 4H), 3.82 (s, 3H),3.83 (s, 3H), 6.48 (s, 1H), 6.58 (s, 1H), 6.83-6.87 (d, 1H), 7.00-7.04(m, 1H), 7.10-7.16 (m, 1H), 7.70-7.90 (s, 1H).

Example 2

5-Bromo-N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-hydroxybenzamide(4b). Following procedure A (see Example 1), 1.16 g (66% yield) of 4bwas obtained. ¹H NMR (300 MHz, CDCl₃): 1.70-1.80 (m, 4H), 2.52-2.62 (m,2H), 2.75-2.84 (m, 4H), 3.44-3.50 (m, 2H), 3.55-3.62 (m, 2H), 3.83 (s,6H), 5.19 (s, 1H), 6.49 (s, 1H), 6.57 (s, 1H), 6.80-6.86 (d, 1H),7.36-7.46 (m, 2H), 8.05-8.08 (s, 1H).

Example 3

N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-hydroxy-5-iodobenzamide(4c). Following procedure A (see Example 1), 1.34 g (69% yield) of 4cwas obtained as a white solid. ¹H NMR (300 MHz, CDCl₃): 1.70-1.80 (m,4H), 2.54-2.62 (m, 2H), 2.76-2.84 (m, 4H), 3.42-3.50 (m, 2H), 3.60 (s,2H), 3.83 (s, 6H), 5.19 (s, 1H), 6.49 (s, 1H), 6.58 (s, 1H), 6.72-6.76(d, 1H), 7.56-7.62 (m, 2H), 8.24-8.26 (s, 1H).

Example 4

5-Bromo-N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-hydroxy-3-methoxybenzamide(4d). Following procedure A, 0.73 mg (22% yield) of 4d was obtained. ¹HNMR (300 MHz, CDCl₃): 1.60-1.80 (m, 4H), 2.50-2.62 (m, 2H), 2.70-2.88(m, 4H), 3.40-3.50 (m, 2H), 3.58 (s, 2H), 3.83 (s, 3H), 3.84 (s, 3H),3.87 (s, 3H), 5.20 (s, 1H), 6.49 (s, 1H), 6.57 (s, 1H), 6.98 (s, 1H),7.10 (s, 1H), 4 (s, 1H).

Example 5

This example demonstrates General procedure B for synthesizing somecompounds of the present teachings.

N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-(2-(2-hydroxyethoxy)ethoxy)-5-methylbenzamide(5a). The mixture of 4a (0.65 g, 1.6 mmol), 2-(2-chloroethoxy)ethanol(0.64 g, 5.1 mmol), potassium carbonate (0.71 g, 5.1 mmol) in ofN,N-Dimethylformamide (DMF) (25 mL) was refluxed over night under anatmosphere of nitrogrn. The reaction mixture was cooled to roomtemperature and quenched with 100 mL water. The aqueous solution wasextracted with ethyl acetate (3 X₂ 50 mL). The combined organic layerwas washed with brine (3×30 mL) and dried over Na₂SO₄. Afterconcentrating under reduced pressure, the residue was purified by silicagel column chromatography with methanol/dichloromethane (10/90, v/v) asthe mobile phase to afford compound 5a (0.41 g; 51% yield) as a viscousyellow oil. ¹H NMR (300 MHz, CDCl₃): 1.68-1.79 (m, 4H), 2.31 (s, 3H),2.50-2.60 (m, 2H), 2.72-2.76 (m, 2H), 2.82-2.85 (m, 2H), 3.46-3.60 (m,8H), 3.80-3.90 (m, 8H), 4.18-4.24 (m, 2H), 6.49 (s, 1H), 6.58 (s, 1H),6.79-6.83 (d, 1H), 7.16-7.24 (m, 1H), 7.98-7.80 (m, 1H), 8.18-8.26 (s,1H).

Example 6

N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)-5-methylbenzamide(5b). Following procedure B (see Example 5), 390 mg (40% yield) of 5bwas obtained. ¹H NMR (300 MHz, CDCl₃): 1.67-1.78 (m, 4H), 2.30 (s, 3H),2.50-2.60 (m, 2H), 2.68-2.74 (m, 2H), 2.76-2.85 (m, 2H), 3.44-3.58 (m,6H), 3.60-3.72 (m, 6H), 3.80-3.90 (m, 8H), 4.16-4.24 (m, 2H), 6.49 (s,1H), 6.57 (s, 1H), 6.80-6.84 (d, 1H), 7.16-7.21 (m, 1H), 7.96-7.80 (m,1H), 8.14-8.24 (s, 1H).

Example 7

5-Bromo-N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-(2-(2-hydroxyethoxy)ethoxy)-benzamide(5c). Following procedure B (see Example 5), 480 mg (70% yield) of 5cwas obtained. ¹H NMR (300 MHz, CDCl₃): 1.70-1.80 (m, 4H), 2.53-2.68 (m,2H), 2.70-2.78 (m, 2H), 2.80-2.88 (m, 2H), 3.44-3.58 (m, 8H), 3.80-3.90(m, 8H), 4.20-4.24 (m, 2H), 6.50 (s, 1H), 6.59 (s, 1H), 6.79-6.82 (d,1H), 7.47-7.51 (m, 1H), 8.10-8.18 (s, 1H), 8.30-8.32 (s, 1H).

Example 8

5-Bromo-N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-(2-(2-(2-hydroxyethoxy)ethoxy)-ethoxy)benzamide(5d). Following procedure B (see Example 5), 420 mg (56% yield) of 5dwas obtained. ¹H NMR (300 MHz, CDCl₁): 1.70-1.80 (m, 4H), 2.53-2.68 (m,2H), 2.70-2.78 (m, 2H), 2.80-2.88 (m, 2H), 3.44-3.58 (m, 6H), 3.60-3.75(m, 6H), 3.80-3.90 (m, 6H), 4.20-4.24 (m, 2H), 6.50 (s, 1H), 6.58 (s,1H), 6.78-6.82 (d, 1H), 7.47-7.51 (m, 1H), 8.10-8.18 (s, 1H), 8.30-8.32(s, 1H).

Example 9

N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-(2-(2-hydroxyethoxy)ethoxy)-5-iodobenzamide(5e). Following procedure B (see Example 5), 360 mg (56% yield) of 5ewas obtained. ¹H NMR (300 MHz, CDCl₃): 1.65-1.80 (m, 4H), 2.50-2.60 (m,2H), 2.70-2.80 (m, 2H), 2.80-2.90 (m, 2H), 3.40-3.60 (m, 8H), 3.68-3.95(m, 8H), 4.18-4.22 (m, 2H), 6.50 (s, 1H), 6.59 (s, 1H), 6.66-6.70 (d,1H), 7.64-7.69 (d, 1H), 8.08-8.16 (s, 1H), 8.40-8.47 (s, 1H).

Example 10

N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-(2-(2-(2-hydroxyethoxy)ethoxy)ethoxy)-5-iodobenzamide(5f). Following procedure B (see Example 5), 650 mg (90% yield) of 5fwas obtained. ¹H NMR (300 MHz, CDCl₃): 1.62-1.80 (m, 4H), 2.50-2.60 (m,2H), 2.68-2.76 (m, 2H), 2.76-2.86 (m, 2H), 3.40-3.56 (m, 6H), 3.58-3.70(m, 6H), 3.70-3.95 (m, 8H), 4.16-4.22 (m, 2H), 6.49 (s, 1H), 6.58 (s,1H), 6.66-6.70 (d, 1H), 7.64-7.68 (m, 1H), 8.04-8.16 (s, 1H), 8.40-8.43(s, 1H).

Example 11

5-Bromo-N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-3-hydroxy-2-(2-(2-hydroxyethoxy)-ethoxy)benzamide(5g). Following procedure B (see Example 5), 0.613 mg (28% yield) of 5gwas obtained. ¹H NMR spectrum (300 MHz, CDCl₃): 1.65-1.80 (m, 4H),2.50-2.60 (m, 2H), 2.70-2.78 (m, 2H), 2.80-2.88 (m, 2H), 3.40-3.65 (m,8H), 3.70-3.75 (m, 2H), 3.83 (s, 3H), 3.84 (s, 3H), 3.85 (s, 1H),4.16-4.22 (m, 2H), 6.50 (s, 1H), 6.58 (s, 1H), 7.08-7.10 (d, 1H),7.82-7.84 (m, 1H), 8.28-8.36 (s, 1H).

Example 12

This example demonstrates General procedure C for synthesizing somecompounds of the present teachings.

N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-(2-(2-fluoroethoxy)ethoxy)-5-methylbenzamide(6a). To solution of compound 5a (0.20 g, 0.41 mmol) in dichloromethane(60 ml) was added slowly (Diethylamino)sulfur trifluoride (DAST) (0.13g, 0.81 mmol) at 0° C. The reaction mixture was stirred overnight underan atmosphere of nitrogen and then quenched with water (30 ml). Theorganic layer was separated and washed with saturated sodium carbonatesolution (2×15 ml) and dried over sodium sulfate. Volatile componentswere removed under reduced pressure. The crude product was purified bysilica gel column chromatography using ether:methanol (100:20) as themobile phase to give 6a (0.07 g; 36% yield) as a viscous colorless oil.¹H NMR spectrum (300 MHz, CDCl₃): 1.65-1.69 (m, 4H), 2.32 (s, 3H),2.50-2.59 (m, 2H), 2.70-2.73 (m, 2H), 2.78-2.82 (m, 2H), 3.45-3.48 (m,2H), 3.54 (s, 2H), 3.69-3.73 (m, 1H), 3.72-3.82 (m, 7H), 3.87-3.90 (m,2H), 4.19-4.22 (m, 2H), 4.43-4.47 (m, 1H), 4.59-4.63 (m, 1H), 6.50 (s,1H), 6.58 (s, 1H), 6.80-6.84 (d, 1H), 7.05-7.20 (m, 1H), 7.97-7.99 (m,1H), 8.21 (s, 1H). The free base was converted into oxalic acid salt andre-crystallized in methanol and ethyl acetate solvent. Mp 89.2-89.55° C.Elemental Analysis: C₂₇H₃₇FN₂O₅.H₂C₂O₄.0.5H₂O (C, H, N). Calcd: C,59.27; H, 6.86; N, 4.77; Found: C, 58.95; H, 6.84; N, 4.85.

Example 13

N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)-5-methylbenzamide(6b). Same as procedure C (see Example 12), 295 mg (76% yield) of 6b wasobtained. ¹H NMR (300 MHz, CDCl₃): 1.60-1.80 (m, 4H); 2.32 (s, 3H),2.50-2.59 (m, 2H), 2.70-2.76(m, 2H), 2.78-2.84 (m, 2H), 3.42-3.50 (m,2H), 3.55 (s, 2H), 3.60-3.80 (m,6H), 3.80-3.90 (m, 8H), 4.19-4.24 (m,2H), 4.43-4.47 (m, 1H), 4.58-4.62 (m, 1H), 6.50 (s, 1H), 6.57 (s, 1H),6.80-6.84 (m, 1H), 7.17-7.23 (d, 1H), 7.96-8.01 (d, 1H), 8.19 (s, 1H).The free base was converted into oxalic acid salt and its melting pointis 127.7-129.0° C. Elemental Analysis: C₂₉H₄₁FN₂O₆.H₂C₂O₄ (C, H, N)Calcd: C, 59.80; H, 6.96; N, 4.50; Found: C, 59.54; H, 7.03; N, 4.57.

Example 14

5-Bromo-N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-(2-(2-fluoroethoxy)ethoxy)benzamide(6c). Same as procedure C (see Example 12), 67 mg (14% yield) of 6c wasobtained as a viscous yellow oil. ¹H NMR spectrum (300 MHz, CDCl₃):1.62-1.75 (m, 4H), 2.52-2.62 (m, 2H), 2.65-2.76 (m, 2H), 2.76-2.82 (m,2H), 3.40-3.51 (m, 2H), 3.56 (s, 2H), 3.70-3.75 (t, 1H), 3.80-3.86 (m,7H), 3.86-3.93 (m, 1H), 4.18-4.23 (t, 2H), 4.42-4.46 (m, 1H), 4.59-4.64(m, 1H), 6.49 (s, 1H), 6.57 (s, 1H), 6.79-6.83 (d, 1H), 7.47-7.52 (m,1H), 8.04-8.14 (s, 1H), 8.27-8.29 (d, 1H). The free base was convertedinto oxalic acid salt (mp: 124.4-126.0° C.). Elemental Analysis:C₂₆H₃₄BrFN₂O₅.H₂C₂O₄.0.5H₂O (C, H, N) Calcd: C, 51.54; H, 5.72; N, 4.29;Found: C, 51.74; H, 5.71; N, 4.21.

Example 15

5-Bromo-N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)benzamide(6d). Following procedure C (see Example 12), 240 mg (58% yield) of 6dwas obtained as a viscous yellow oil. ¹H NMR (300 MHz, CDCl₃): 1.60-1.80(m, 4H), 2.52-2.60 (m, 2H), 2.65-2.76 (m, 2H), 2.76-2.84 (m, 2H),3.42-3.52 (m, 2H), 3.55 (s, 2H), 3.60-3.74 (m, 6H), 3.82 (s, 3H), 3.83(s, 3H), 3.86-3.90 (m, 2H), 4.18-4.22 (m, 2H), 4.42-4.46 (m, 1H),4.58-4.62 (m, 1H), 6.49 (s, 1H), 6.57 (s, 1H), 6.79-6.83 (d, 1H),7.46-7.52 (m, 1H), 8.06-8.15 (s, 1H), 8.26-8.30 (d, 1H). The free basewas converted into oxalic acid salt (mp: 89.5-91.0° C.) ElementalAnalysis: C₂₈H₃₈BrFN₂O₆.H₂C₂O₄.0.5H₂O (C, H, N) Calcd: C, 51.73; H,5.93; N, 4.02; Found: C, 51.58; H, 5.89; N, 4.06.

Example 16

N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)-5-iodobenzamide(6e). Following procedure C (see Example 12), 170 mg of 6e (47% yield)was obtained as viscous brown oil. ¹H NMR (300 MHz, CDCl₃): 1.60-1.70(m, 4H), 2.52-2.60 (m, 2H), 2.64-2.74 (t, 2H), 2.76-2.84 (t, 2H),3.40-3.49 (m, 2H), 3.54 (s, 2H), 3.68-3.73 (t, 1H), 3.73-3.85 (m, 7H),3.85-3.92 (m, 2H), 4.18-4.22 (m, 2H), 4.40-4.46 (m, 1H), 4.58-4.62 (1H),6.49 (s, 1H), 6.57 (s, 1H), 6.66-6.72 (d, 1H), 7.64-7.70 (m, 1H),8.00-8.10 (s, 1H), 8.44-8.46 (d, 1H). The free base was converted intooxalic acid salt (mp: 112.0-114.0° C.). Elemental Analysis results:C₂₆H₃₄FIN₂O₅.H₂C₂O₄ (C, H, N) Calcd: C, 48.70; H, 5.26; N, 4.06; Found:C, 48.40; H, 5.34; N, 3.92.

Example 17

N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)-5-iodobenzamide(6f). Following procedure C (see Example 12), 360 mg of 6f (56% yield)was obtained as a viscous brown oil. ¹H NMR (300 MHz, CDCl₃): 1.60-1.75(m, 4H), 2.48-2.60 (m, 2H), 2.70-2.72 (t, 2H), 2.77-2.82 (t, 2H),3.40-3.49 (m, 2H), 3.53 (s, 2H), 3.60-3.76 (m, 6H), 3.82 (s, 3H), 3.83(s, 3H), 3.85-3.89 (m, 2H), 4.17-4.23 (m, 2H), 4.41-4.45 (m, 1H),4.57-4.61 (m, 1H), 6.49 (s, 1H), 6.57 (s, 1H), 6.67-6.72 (d, 1H),7.62-7.68 (m, 1H), 8.02-8.12 (s, 1H), 8.43-8.45 (d, 1H). The free basewas converted into oxalic acid salt (mp: 92.0-93.5° C.). ElementalAnalysis: C₂₈H₃₈FIN₂O₆.H₂C₂O₄.0.5H₂O (C, H, N) Calcd: C, 48.46; H, 5.56;N, 3.77; Found: C, 48.51; H, 5.41; N, 3.78.

Example 18

5-bromo-N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-(2-(2-fluoroethoxy)ethoxy)-3-methoxybenzamide(6g). Following procedure C (see Example 12), 360 mg (16% yield) of 6gwas obtained as a viscous brown oil. ¹H NMR (300 MHz, CDCl₃): 1.60-1.78(m, 4H), 2.48-2.63 (m, 2H), 2.66-2.75 (m, 2H), 2.76-2.85 (m, 2H),3.40-3.51 (m, 2H), 3.57 (s, 2H), 3.68-3.73 (m, 1H), 3.72-3.80 (m, 12H),4.16-4.29 (m, 2H), 4.44-4.50 (m, 1H), 4.60-4.67 (m, 1H), 6.51 (s, 1H),6.58 (s, 1H), 7.09-7.12 (d, 1H), 7.85-7.88 (d, 1H), 8.30-8.40 (s, 1H).The free base was converted into oxalic acid salt (mp: 116.0-118.0° C.).Elemental Analysis: C₂₇H₃₆BrFN₂O₆.H₂C₂O₄.H₂O (C, H, N) Calcd: C, 50.37;H, 5.83; N, 4.05; Found: 50.58, 5.64; 4.16.

Example 19

This example illustrates synthetic steps for generating ¹⁸F-taggedcompounds of the present teachings.

Example of radiosynthesis of [¹⁸F] compounds. ˜180 mCi [¹⁸F]fluoride canbe dried by azeotropic distillation using CH₃CN (3×1 mL) in the presenceof K₂CO₃ (0.75 mg) and K₂₂₂ (5 mg) at 110° C. under a flow of N₂, then asolution of a compound to be radiolabeled (2.5 mg) in CH₃CN (400 μL) canbe added. K₂₂₂ is4,7,13,16,21,24-Hexaoxa-1,10-diazabicyclo[8.8.8]-hexacosane (Kryptofix222®, Acros Organics N.V., Fairlawn, N.J.). The reaction mixture can beheated in an oil bath (110° C.) for 10 min [incorporation can be, e.g.,17.3±4.4% (n=10) according to radio-TLC analysis: silica, 1:1 ethylacetate/hexanes]. The mixture can then be passed through a silica gelSep-Pak® (Waters) and CH₃CN (2×1 mL) can be used to rinse the reactionvial and the Sep-Pak®. The elution can be concentrated to less than 500μL in the presence of 1N HCl (100 μL) at 110° C. under a flow of N₂,then 1N HCl (500 μL) can be added. The reaction mixture can beirradiated under microwave for 30 and 25 sec with an interval of 30 secbetween each irradiation, and then can be diluted in water (3 mL) forHPLC injection. An [¹⁸F] radiolabeled compound can be purified byreversed phased HPLC using an Alltech® Platinum EPS C18 column (250×10mm, 10μ) eluted with 15% CH₃CN, 85% water with 0.1% trifluoroacetic acid(TFA) at a flow rate of 4 mL/min and UV at 272 nm. The radioactivity (˜8mCi) corresponding to a [¹⁸F] radiolabeled compound can be collected at,e.g., 17 min, and the collection fraction can be concentrated underreduced pressure to less than 0.5 mL and then diluted in water (40 mL).An [¹⁸F] radiolabeled compound can be separated from the dilution bypassing the dilution through an Oasis® HLC cartridge (Waters) and can beeluted from the cartridge with ethanol (1˜2 mL). If necessary, theethanol solution can be concentrated under a flow of N₂ in order to makea final dose, e.g., for animal study with <10% ethanol in saline. Thetotal synthesis and purification time can be 120 min; decay-correctedradiochemical yield can be, e.g., 6.2±2.1%; Radiochemical purity canbe >99.9% and specific activity can be, e.g., 2,160±1,660 mCi/μmol atthe end of synthesis, and can be analyzed using an analytical HPLCcolumn (Alltech® Platinum™ EPS C18 250×4.6 mm, 10μ, 20% CH₃CH, 80%water, 0.1% TFA, 2 mL/min, 272 nm) and determined by comparison of theintegrated UV absorbance with a calibrated mass/UV absorbance curve. Theidentity of a [¹⁸F] radiolabeled compound can be confirmed by thecoelution of the [¹⁸F] radiolabeled compound with a nonradioactivestandard on the analytical HPLC system.

Example 20

This example illustrates synthetic steps for generating ¹⁸F-taggedcompounds of the present teachings.

Example of radiosynthesis of [¹⁸F] compounds. [¹⁸F]fluoride (100-150mCi) can be added to a 10-mL Pyrex screw cap tube containing 5-6 mg ofKryptofix 222® and 0.75 mg of K₂CO₃. Using HPLC grade acetonitrile(3×1.0 mL), the water can be azeotropically evaporated from this mixtureat 110° C. under a stream of argon. After all of the water is removed, asolution of a precursor (1.5-2.0 mg) in DMSO (0.2 mL) can be added tothe reaction vessel containing the ¹⁸F/Kryptofix mixture. A 3 mm glassbead can be added to the reaction vessel to insure a more homogeneousheat distribution when the sample is irradiated with microwaves, and thevessel can be capped firmly on a remotely operated capping station.After vortexing, the reaction mixture can be irradiated with microwavesfor 30-40 sec at medium power (60 Watts) until the thin layerchromatography scanner with a 25% of methanol and 75% dichloromethanemobile phase indicates that the incorporation yield can be 40-60%.

After adding 6 mL of water and shaking, the solution can be loaded on aC-18 reverse phase Waters Oasis® cartridge (HLB-6 cc) that haspreviously been rinsed with a solution of 5% methanol in water (5-8 mL).The sample can then be rinsed 3 times with 6 mL water to eliminate theunreacted fluoride. The retained activity can be eluted with 5-8 mL ofacetonitrile. After evaporating the acetonitrile to a volume of <0.5 mL,the sample can be loaded on a C-18 Alltech® econosil semi-preparativeHPLC column (250×10 um). The product can be eluted with 29% acetonitrileand 71% 0.1M ammonium formate buffer at a flow rate of 4.5 mL/min. Theretention time of the [¹⁸F] radiolabeled compound can be ˜33 min. Thesolution containing the [¹⁸F] radiolabeled compound can be concentrated,resuspended in saline, and a 100 uL aliquot sent for quality controlanalysis before using it in the biodistribution and imaging studies. Theentire procedure requires ˜2 h.

Quality control analysis can be performed on an analytical HPLC systemthat can consist of an Alltech® econosil reversed phase C-18 column(250×4.6 mm) with a mobile phase of 35% acetonitrile and 65% 0.1 Mammonium formate buffer at pH 4.0-4.5. At a flow rate of 1.2 mL/min, the[¹⁸F] radiolabeled compound can elute at, e.g., 13.2 min with aradiochemical purity of >99%. The labeling yield can be ˜30% (decaycorrected), and the specific activity can be >2000 Ci/mmol.

Example 21

This example illustrates in vitro binding studies with the compounds ofthe present teachings. In this example, in vitro binding studies can beconducted in order to measure the affinity of the target compounds forσ₁ and σ₂ receptors.

In these assays, pegylated sigma ligands of the present teachings can bedissolved in N,N-dimethylformamide (DMF), DMSO or ethanol, and can thenbe diluted in 50 mM Tris-HCl buffer containing 150 mM NaCl and 100 mMEDTA at pH 7.4 prior to performing σ₁ and σ₂ receptor binding assaysProcedures for isolating the membrane homogenates and performing the σ₁and σ₂ receptor binding assays have been described in detail previously(Xu, J., et al., Eur. J. Pharmacol. 21: 525 (1-3): 8-17, 2005). Briefly,the σ₁ receptor binding assays can be conducted in 96-well plates usingguinea pig brain membrane homogenates (˜300 μg protein) and ˜5 nM[³H](+)-pentazocine (34.9 Ci/mmol, Perkin Elmer, Boston, Mass.). Thetotal incubation time can be 90 min at room temperature. Nonspecificbinding can be determined from samples that contain 10 μM of coldhaloperidol. After 90 min, the reaction can be terminated by theaddition of 150 μL of ice-cold wash buffer (10 mM Tris-HCl, 150 mM NaCl,pH 7.4) using a 96 channel transfer pipette (Fisher Scientific,Pittsburgh, Pa.). The samples can be harvested and filtered rapidlythrough a 96-well fiber glass filter plate (Millipore, Billerica, Mass.)that has been presoaked with 100 μL of 50 mM Tris-HCl buffer at pH 8.0for 1 h. Each filter can be washed 3 times with 200 μL of ice-cold washbuffer, and the filter can be counted in a Wallac 1450 MicroBeta liquidscintillation counter (Perkin Elmer, Boston, Mass.).

σ₂ receptor binding assays can be conducted using rat liver membranehomogenates (˜300 μg protein) and ˜5 nM [³H]DTG (58.1 Ci/mmol, PerkinElmer, Boston, Mass.) in the presence of 1 μM (+)-pentazocine. Theincubation time can be 120 mM at room temperature. Nonspecific bindingcan be determined from samples that contain 10 μM of cold haloperidol.All other procedures can be identical to those described for the σ₁receptor binding assay above.

Data from the competitive inhibition experiments can be modeled usingnonlinear regression analysis to determine the concentration thatinhibits 50% of the specific binding of the radioligand (IC₅₀ value).Competitive curves can be best fit to a one-site fit and givepseudo-Hill coefficients of 0.6-1.0. K_(i) values can be calculatedusing the method of Cheng and Prusoff (Biochem. Pharmacol. 22:3099-3108, 1973) and can be presented as the mean±1 SEM. For thesecalculations, one can use a K_(d) value of 7.89 nM for[³H](+)-pentazocine and guinea pig brain; for [³H]DTG and rat liver, forexample 30.73 nM.

TABLE 1 Pegylated Compounds # Structure Formula 6a

C₂₇H₃₇FN₂O₅ (488.59) 6b

C₂₉H₄₁FN₂O₆ (532.64) 6c

C₂₆H₃₄BrFN₂O₅ (553.46) 6d

C₂₈H₃₈BrFN₂O₆ (597.51) 6e

C₂₆H₃₄FIN₂O₅ (600.46) 6f

C₂₈H₃₈FIN₂O₆ (644.51) 6g

C₂₇H₃₆BrFN₂O₆ (583.49) 6h

C₂₉H₄₀BrFN₂O₇ (627.54)

TABLE 2 σ₁ and σ₂ binding affinities (K_(i)) and log D of pegylatedligands

# X₁ X₂ m σ₁ σ₂ Ratio (σ1/σ2) Log D^(a) 6a H CH₃ 2  277 ± 38 6.75 ± 0.9 41 2.66 6b H CH₃ 3 1,922 ± 238 10.09 ± 0.4  190 2.30 6c H Br 2 1,043 ±151 1.38 ± 0.2 755 3.49 6d H Br 3 1,013 ± 171 9.80 ± 0.4 103 3.13 6e H I2  451 ± 56 6.29 ± 0.6  72 3.74 6f H I 3 1,278 ± 51  7.47 ± 1.2 171 3.386g OCH₃ Br 2   669 ± 101 4.67 ± 0.6 143 3.16 6h OCH₃ Br 3 9097 33 276 ND^(a)calculated using the program ACD/log D

Example 22

This example illustrates in vivo evaluation of compounds of the presentteachings. All animal experiments can be conducted in compliance withthe Guidelines for the Care and Use of Research Animals established byWashington University's Animal Studies Committee. EMT-6 mouse mammaryadenocarcinoma cells (5×10⁵ cells in 100 uL of phosphate-bufferedsaline) can be implanted subcutaneously in the scapular region of femaleBalb/c mice (˜2-month old and 17-22 g; Charles River Laboratories). Thebiodistribution studies can be initiated 7-10 days after implantationwhen the tumor size can be ˜0.2 cm³ (˜200 mg).

For biodistribution studies, 10-120 μCi of an [¹⁸F]-radiolabeledpegylated compound of the present teachings in 100-150 uL of saline canbe injected via the tail vein into EMT-6 tumor-bearing female Balb/cmice. Groups of at least 4 mice can be used for each time point. At 5,30, 60, and 120 min after injection, the mice can be euthanized, andsamples of blood, lung, liver, kidney, muscle, fat, heart, brain, boneand tumor can be removed, weighed and counted in a Beckman Gamma 8000well counter. After counting, the percentage of the injected dose pergram of tissue (% ID/g) can be calculated. The tumor/organ ratios can becalculated by dividing the % ID/g of the tumor by the % ID/g of eachorgan.

Labeled compounds can display tumor uptake at 5 min post-injection.Tumor uptake at 1 h post-injection can remain high for each of theligands, and can continue to remain relatively high at 2 hpost-injection in comparison to that of the normal tissues, fat andmuscle. This can result in acceptable tumor:normal tissue ratios for PETimaging. Also, a low bone uptake of a labeled compound, which cancontinue to decrease between 30 min and 1 h time points, can suggestthat these compounds do not undergo a significant defluorination invivo.

Example 23

This example illustrates specificity of binding in vivo for σ₂ receptorsby pegylated compounds of the present teachings.

To demonstrate that the in vivo binding of radiolabeled compound of thepresent teachings can be specific for σ₂ receptors, a no-carrier-addeddose of these radiotracers can be co-injected into EMT-6 tumor-bearingmice with N-(4-fluorobenzyl)piperidinyl-4-(3-bromophenyl)acetamide(YUN-143), a sigma ligand displaying a high affinity for both σ₁ and σ₂receptors. Co-injection of YUN-143 with a pegylated compound of thepresent teachings can result in a decrease in the tumor:muscle andtumor:fat ratios at 1 h post-injection.

Blocking studies in tumor-bearing mice can be conducted by co-injecting1 mg/kg of cold N-(4-fluorobenzyl)piperidinyl-4-(3-bromophenyl)acetamide(YUN-143) with a pegylated compound of the present teachings. Yun-143has a high affinity for both σ₁ and σ₂ receptors and is routinely usedfor sigma receptor blocking studies (Mach, R. H., et al., Nucl Med.Biol. 28: 451-458, 2001; Bowen, W. D. et al., Eur. J. Pharmacol. 278:257-260, 1995; Bonhaus, D. W. et al., J. Pharmacol. Exp. Ther. 267: 961,1993. All mice can be sacrificed 60 min after injection of theradiotracer, and the tumor:organ ratios can be determined as describedabove.

Example 24

This example illustrates use of radioligands of the present teachings asimaging agents.

To demonstrate feasibility of using radioligands of the presentteachings as PET imaging agents for determining the σ₂ receptor statusof solid tumors, a CT/PET study using a compound of the presentteachings in female Balb/c mice bearing EMT-6 tumors can be performed ona microPET-F220 (CTI-Concorde Microsystems Inc.) and a MicroCAT-IIsystem (ImTek Inc.). For the microPET studies, each mouse can beinjected with ˜0.25 mCi of a compound of the present teachings via thetail vein and imaged 1 h later. MicroCT images can also be obtained andco-registered with the PET images to determine the exact anatomicallocation of the radiotracers.

EMT-6 tumors can be identified using a radioligand. Such studies couldindicate that a compound of the present teachings can be an acceptableagent for detecting solid tumors and imaging their σ₂ receptor statuswith PET.

Example 25

This example illustrates production of [18F]Fluoride.

[¹⁸F]Fluoride was produced in our institution by proton irradiation ofenriched ¹⁸O water (95%) [reaction: ¹⁸O(p, n)¹⁸F] using either a JSWBC-16/8 (Japan Steel Works) or a CS-15 cyclotron (Cyclotron Corp).

Example 26

This example illustrates tumor uptake of ¹⁸F-labeled pegylated compounds6c, 6d and 6g versus a nonpegylated analog (m=1; X₁=H; X₂=Br).

In these experiments, various ¹⁸F-labeled pegylated compounds of thepresent teachings as well as a non-pegylated analog were administered totumor-bearing mice as described in Example 22. Biodistribution of thecompounds were analyzed; results are presented in FIG. 2. The pegylatedcompounds tested,

were 6c (m=2; X₁=H; X₂=Br), 6d (m=3; X₁=H; X₂=Br) and 6g (m=2; X₁=OCH₃;X₂=Br). A non-pegylated analog (m=1; X₁=H; X₂=Br) was also tested. Thedata demonstrate higher tumor uptake compared to the non-pegylatedanalog. The data also demonstrate a slower rate of washout of thepegylated analogs versus the non-pegylated analog.

All patents and publications cited herein are hereby incorporated byreference, each in its entirety.

What is claimed is:
 1. A radiolabeled pegylated fluoroalkoxybenzamidecompound or salt thereof of structure

wherein m is an integer from 2 to 5, n is an integer from 1 to 10, andX₁ and X₂ are each independently selected from the group consisting ofH, a halogen selected from the group consisting of I, Br, Cl and F, aC₁₋₄ alkoxy, a C₁₋₄ alkyl, a C₁₋₄ fluoroalkyl, a C₁₋₄ fluoroalkoxy, CF₃,OCF₃, SCH₃, SCF₃, and NH₂.
 2. A radiolabeled pegylatedfluoroalkoxybenzamide compound or salt thereof in accordance with claim1, wherein m=2, n=4, X₁ is H, and X₂ is CH₃.
 3. A radiolabeled pegylatedfluoroalkoxybenzamide compound or salt thereof in accordance with claim1, wherein m=3, n=4, X₁ is H, and X₂ is CH₃.
 4. A radiolabeled pegylatedfluoroalkoxybenzamide compound or salt thereof in accordance with claim1, wherein m=2, n=4, X₁ is H, and X₂ is Br.
 5. A radiolabeled pegylatedfluoroalkoxybenzamide compound or salt thereof in accordance with claim1, wherein m=3, n=4, X₁ is H, and X₂ is Br.
 6. A radiolabeled pegylatedfluoroalkoxybenzamide compound or salt thereof in accordance with claim1, wherein m=2, n=4, X₁ is H, and X₂ is I.
 7. A radiolabeled pegylatedfluoroalkoxybenzamide compound or salt thereof in accordance with claim1, wherein m=3, n=4, X₁ is H, and X₂ is I.
 8. A radiolabeled pegylatedfluoroalkoxybenzamide compound or salt thereof in accordance with claim1, wherein m=2, n=4, X₁ is methoxy, and X₂ is Br.
 9. A radiolabeledpegylated fluoroalkoxybenzamide compound or salt thereof in accordancewith claim 1, wherein m=3, n=4, X₁ is methoxy, and X₂ is Br.
 10. Aradiolabeled pegylated fluoroalkoxybenzamide compound or salt thereof ofstructure

wherein m is an integer from 2 to 5, n is an integer from 1 to about 10,and X₁ is selected from the group consisting of H, a halogen selectedfrom the group consisting of I, Br, Cl and F, a C₁₋₄ alkoxy, a C₁₋₄alkyl, a C₁₋₄ fluoroalkyl, a C₁₋₄ fluoroalkoxy, CF₃, OCF₃, SCH₃, SCF₃,and NH₂ and X₂ is selected from the group consisting of ⁷⁶Br, ¹²³I and¹²⁴I.
 11. A radiolabeled pegylated fluoroalkoxybenzamide compound orsalt thereof in accordance with claim 10, wherein m=2, n=4, X₁ is H, andX₂ is ⁷⁶Br.
 12. A radiolabeled pegylated fluoroalkoxybenzamide compoundor salt thereof in accordance with claim 10, wherein m=3, n=4, X₁ is H,and X₂ is ⁷⁶Br.
 13. A radiolabeled pegylated fluoroalkoxybenzamidecompound or salt thereof in accordance with claim 10, wherein m=2, n=4,X₁ is H, and X₂ is ¹²³I or ¹²⁴I.
 14. A radiolabeled pegylatedfluoroalkoxybenzamide compound or salt thereof in accordance with claim10, wherein m=3, n=4, X₁ is H, and X₂ is ¹²³I or ¹²⁴I.
 15. Aradiolabeled pegylated fluoroalkoxybenzamide compound or salt thereof inaccordance with claim 10, wherein m=2, n=4, X₁ is methoxy, and X₂ is⁷⁶Br.
 16. A radiolabeled pegylated fluoroalkoxybenzamide compound orsalt thereof in accordance with claim 10, wherein m=3, n=4, X₁ ismethoxy, and X₂ is ⁷⁶Br.
 17. A method of imaging a tumor in a mammal,the method comprising: administering to the mammal a radiolabelledpegylated fluoroalkoxybenzamide compound of claim 1 or a salt thereof;and subjecting the mammal to positron emission tomography (PET)scanning.
 18. A method of imaging a tumor in accordance with claim 17,wherein m=2, n=4, X₁ is H, and X₂ is Br.
 19. A method of imaging a tumorin accordance with claim 17, wherein m=3, n=4, X₁ is methoxy, and X₂ isBr.
 20. A method of imaging a tumor in accordance with claim 17, whereinm=3, n=4, X₁ is H, and X₂ is CH₃.